Abstract Duchenne muscular dystrophy (DMD) is a genetic disease caused by mutations in the dystrophin gene and causes thousands of deaths each year. There are no effective treatments for DMD and new DMD therapeutics are urgently needed. Cas9 based therapeutics have the potential to revolutionize the treatment of DMD, because they can correct dystrophin mutations, via homology directed DNA repair. However, developing Cas9 based therapeutics for DMD has been challenging because it requires simultaneously delivering Cas9 protein, guide RNA, and donor DNA in vivo and delivery vehicles have not been developed that can accomplish this. The central objective of this proposal is to develop a new family of nanoparticle delivery vehicles, termed CRISPR-Nanoparticles, which are designed to treat DMD by delivering Cas9 protein, guide RNA and donor DNA in vivo. CRISPR-Gold is our first generation CRISPR-Nanoparticle and is composed of gold nanoparticles complexed with Cas9 RNP, donor DNA and the endosomal disruptive polymer PASp(DET). We have been able to demonstrate that CRISPR-Gold, can correct 5% of the dystrophin mutations (via HDR) in muscle fibers after a direct injection in mdx mice, and thus has tremendous potential as a treatment for DMD. A successful DMD therapeutic needs to be able to correct 20% of the dystrophin mutations in muscle tissue, and the experiments in this proposal focus on developing 2nd and 3rd generation CRISPR-Nanoparticles, which can generate a 20% HDR rate in muscle tissue. In particular, the 2nd and 3rd generation CRISPR- Nanoparticles address the key factors preventing CRISPR-Gold from generating a 20% HDR rate in a clinical setting, which are (1) its lack of biodegradability, (2) its toxicity, and (3) the lack of cell division in muscle tissue. The central hypothesis of this proposal is that: Delivery vehicles that complex Cas9 protein, guide RNA, donor DNA and endosomal disruptive polymers will be able to efficiently induce HDR and treat DMD. The central objective of this proposal will be accomplished by completing the following Specific Aims. Specific Aim 1: Develop biodegradable CRISPR-Nanoparticles that can correct dystrophin mutations Specific Aim 2: Develop biocompatible CRISPR-Nanoparticles that can correct dystrophin mutations Specific Aim 3: Enhance the HDR efficiency of CRISPR-Nanoparticles with FDA approved stimulating agents At the conclusion of this proposal we will have identified a CRISPR-Nanoparticle formulation that can efficiently correct dystrophin mutations in vivo and has the biocompatibility needed for clinical translation. The experiments in this proposal are innovative because CRISPR-Gold is the first example of a delivery vehicle that can simultaneously deliver RNA, DNA and protein in vivo and induce HDR. The experiments in this proposal are significant because they will lead to the development of a new therapeutic for DMD (and other genetic diseases) that has the HDR efficiency and biocompatibility needed to enter into clinical trials.